Preparation method of Maillard reaction modified corn peptide loaded xanthophyll nanoparticles

Abstract

According to the invention, the problems of complex preparation process, low embedding transmission efficiency, poor dissolution and dispersibility of xanthophyll, low absorption and utilization rate and the like of the xanthophyll embedding carrier are solved. The preparation method of the Maillard reaction modified corn peptide loaded xanthophyll nanoparticles comprises the following steps: 1, preparing a chitosan oligosaccharide solution and a corn peptide solution; 2, adjusting the pH value of the solution; 3, preparing glycosylated corn peptide through Maillard reaction; 4, carrying out load combination on the glycosylated corn peptide and the xanthophyll; and 5, preparing the xanthophyll nanoparticles. The method has the advantages that 1, the embedding rate and the solubility of the lutein are effectively improved; 2, the stability and the dispersity of the xanthophyll nanoparticles are improved; the absorption and utilization rate is good, the nutritive value is high, and large-scale production is easy. The preparation method of the xanthophyll nanoparticles is simple in process and good in xanthophyll controlled release effect and has a targeted absorption function.

Description

Technical Field

[0001] This invention relates to a method for preparing Maillard reaction modified corn peptide-loaded lutein nanoparticles. Background Technology

[0002] Lutein (LUT) is a naturally occurring oxygenated carotenoid, also known as "plant lutein," widely found in green vegetables, corn, egg yolks, and other foods. Carotenoids absorb wavelengths from 400 to 550 nm and can be broadly classified into anaerobic and oxygenated carotenoids. Oxygenated carotenoids are called lutein, such as zeaxanthin. As an important antioxidant, lutein plays an indispensable role in protecting vision health. The human body cannot synthesize lutein and must obtain it from the diet, primarily from egg yolks and leafy green vegetables. Numerous basic and clinical studies have reported the antioxidant and anti-inflammatory properties of lutein in the eye, indicating its positive effects in preventing eye diseases such as macular degeneration, diabetic retinopathy, retinopathy of prematurity, myopia, and cataracts. Lutein can also inhibit the growth of human tumor cells and may help prevent various cancers, such as skin cancer, breast cancer, and colon cancer. However, like other carotenoids, lutein is easily degraded by the conjugated double bonds in its structure under adverse environments such as strong acids, strong alkalis, and high temperatures, resulting in discoloration and inactivation, which severely limits its industrial application. The low water solubility of lutein is another major challenge limiting its application. As a fat-soluble component, lutein can only dissolve in organic solvents. After ingestion, lutein needs to be digested in the gastrointestinal tract, finally dissolved and absorbed in the small intestine, and transported to the bloodstream via membrane proteins. However, its poor water solubility makes it difficult for lutein to be absorbed by the small intestinal epithelial cells, resulting in low bioavailability.

[0003] Corn peptides (CPTs) are bioactive peptides derived from corn protein through enzymatic or acid-base hydrolysis and purification processes. Composed of short-chain amino acid residues, their small molecular structure allows for absorption in the human body at a much faster rate than that of large protein molecules. This not only preserves the basic nutritional components of corn protein but also possesses strong physiological activity, primarily manifested in antioxidation, blood pressure reduction, and immune enhancement. Studies have shown that corn peptides have the ability to scavenge free radicals, effectively resisting oxidative stress and thus playing a role in anti-aging and cell protection. Furthermore, corn peptides can lower blood pressure by affecting the activity of angiotensin-converting enzyme (ACE), making them a potential natural antihypertensive agent for hypertensive patients. Due to their excellent solubility and stability, corn peptides also have wide applications in baking, dairy products, and meat products. With the increasing consumer demand for healthy foods, corn peptides, as a natural and healthy nutrient, have enormous market potential. Simultaneously, researchers are continuously studying the extraction processes, structural characteristics, and new physiological activities of corn peptides, further expanding their applications in the food and pharmaceutical fields. Corn peptides not only have significant nutritional value but also broad application prospects in the food industry.

[0004] Nanotechnology, as a cutting-edge scientific technology, is gradually integrating into all aspects of the food industry, bringing revolutionary changes to food production, processing, packaging, and preservation. Nanoparticles are delivery systems that encapsulate active ingredients or drugs using natural or synthetic polymer materials as carriers, offering advantages such as small size and high stability. Encapsulating unstable active ingredients using nanocarriers can improve their water solubility and stability, reducing losses during processing and storage. Currently, research has been conducted on using corn peptides and their modified products as carriers for active substances. For example, CN114128886A discloses a method to improve the loading rate of curcumin by corn peptides. This method involves mixing a corn peptide solution with a soybean soluble polysaccharide solution, adding transglutaminase for glycosylation, and then encapsulating curcumin to obtain a corn peptide-based curcumin composite nanoparticle solution. Although this invention improves the binding capacity of corn peptides to curcumin and the loading rate of curcumin by cross-linking corn protein peptides using TG enzyme, the enzymatic reaction process is complex and difficult to control, and the polysaccharide composition is relatively complex. Furthermore, the binding and encapsulation mechanism has not been thoroughly elucidated, and the method is limited to mixed binding. Maillard reactions can promote the binding of peptides to sugars and improve their functional properties. Corn peptide Maillard-modified nanoparticles have gradually attracted attention due to their advantages such as simple preparation methods, strong amphiphilicity, high dispersibility and solubility, good biocompatibility, and good targeting. For example, Chinese patent 103445282A discloses a method for preparing corn peptide glycosylated product nanoparticles encapsulating fat-soluble vitamins. This method prepares corn peptide glycosylated product nanoparticles loaded with fat-soluble vitamins, achieving high encapsulation efficiency and small particle size for the fat-soluble vitamins, and the solution exhibits good transparency and stability. However, the dry-heat method used in this method makes it difficult to control the structural properties of the glycosylated products, resulting in weak transport affinity for glycosylated proteins and unclear structure-activity relationships. It is limited to encapsulating liquid, oil-soluble vitamins such as D and E.

[0005] Currently, research on the modification of corn peptides mainly focuses on their chelation with metal ions or the development of physiological activities. However, reports on the application of glycosylated corn peptides as carrier components in functional foods or the medical field are limited. This limitation is due to factors such as poor water solubility hindering the development and utilization of corn peptides as functional foods, especially the limited research on using Maillard reactions to obtain glycosylated corn peptides as carriers. Chitosan oligosaccharide (COS) is a cationic polysaccharide with good stability and high bioavailability. It can bind to the intestinal mucus layer and negatively charged groups of epithelial cells through positively charged amino groups, enhancing mucosal adhesion and barrier permeability, thus facilitating the transmembrane transport and absorption of nutrients. While it has achieved some success in modifying functional carriers and promoting absorption, research on the preparation of highly efficient lutein-carrying nanoparticles using Maillard glycosylation modification of corn peptides has not yet been reported. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing lutein nanoparticles using Maillard-glycosylated modified corn peptides as a carrier, which exhibit good lutein carrying capacity, high solubility, and strong absorption and release properties. This invention solves the problems of low lutein encapsulation efficiency, poor solubility and dispersibility, and low absorption and utilization rate.

[0007] The present invention is achieved through the following technical solutions: 1. The present invention provides a method for preparing Maillard reaction modified corn peptide loaded with lutein nanoparticles, comprising the following steps: (1) Preparation of a mixed solution of chitosan oligosaccharide and corn peptide: Chitosan oligosaccharide with a molecular weight of 1000-3000 and corn peptide with a molecular weight of less than 1000 are dissolved in 100-200 times 70% ethanol at a ratio of (0.5-1.5):1, and the solution is vortexed to dissolve completely; (2) Adjusting the pH of the solution: using 0.4-1.0 mol·L -1 (2) Adjust the pH of the mixed chitosan oligosaccharide and corn peptide solution to 9.0-13.0 with NaOH solution; (3) Prepare glycosylated corn peptide by Maillard reaction: Place the above mixed solution in a 60-90℃ water bath for 60-120 min, cool to room temperature after the reaction, add deionized water to the original volume, adjust the pH of the solution to 7.5-8.0, stir magnetically for 10-30 min to obtain glycosylated corn peptide solution; (4) Combine glycosylated corn peptide with lutein loading: Add an equal volume of lutein standard solution with a concentration of 100μg / mL to the above glycosylated corn peptide solution, and heat at a temperature of 4 Hydrate for 10-30 min at 0-50℃; (5) Preparation of lutein nanoparticles: The above glycosylated corn peptide-lutein solution is concentrated by rotary evaporation at a vacuum of -0.08 to -0.1 MPa and a temperature of 40-50℃ to remove ethanol, and Maillard glycosylated corn peptide-loaded lutein nanoparticles (GCPT-LUT) are obtained; (6) The average particle size of the glycosylated corn peptide-loaded lutein nanoparticles obtained in this invention is mainly concentrated in 200-400 nm, the lutein loading rate can reach more than 91.5%, the polydispersity index PDI≦0.149, and it has good dispersibility and stability.

[0008] 2. Compared with the prior art, the present invention has the following advantages: (1) Compared with other modification techniques, the Maillard reaction method for modifying proteins is relatively simple, the sugar and protein are fully bound, there are few side reactions, it can be used as a carrier for lutein encapsulation and delivery, and the production cost is low, making it easy to scale up and industrialize. (2) The glycosylated corn peptide-loaded lutein nanoparticles prepared by the present invention also showed a high encapsulation rate, which reached more than 90% after optimization. This proves that the glycosylated corn peptide-loaded lutein nanoparticles present a complete encapsulation structure, which can effectively protect the active ingredients, and at the same time improve the stability and bioavailability of lutein in the corn peptide delivery system. (3) The present invention uses the Maillard reaction and vacuum antisolvent method to combine lutein with glycosylated corn peptide carriers to prepare glycosylated corn peptide-lutein nanoparticles. The obtained nanoparticles have a small particle size range (<400nm), exhibiting extremely small particle size, polydispersity index (PDI<0.15) and stable potential, indicating that its particle size distribution is extremely uniform and it is not easy to aggregate, with good stability. (4) The results of transmission electron microscopy showed that there was no adhesion between the particles, and the particle size distribution was uniform and the dispersibility was excellent. This ensured the stability of the glycosylated corn peptide lutein nanoparticles and the integrity of the nanoparticle structure in practical applications, and provided a new approach for the development of lutein nanodelivery systems.

[0009] Attached image description: Figure 1 Distribution of different nanoparticle sizes. Figure 2 Different polydispersity index (PDI) values ​​of nanoparticles. Figure 3 Zeta potential of different nanoparticles. Figure 4 TEM images of different nanoparticles. Figure 5 Infrared spectra of LUT, CPT, COS and GCPT-LUT. Detailed Implementation

[0011] Specific Implementation Method 1: A method for preparing Maillard reaction modified corn peptide-loaded lutein nanoparticles is achieved through the following steps: (1) Preparation of a mixed solution of chitosan oligosaccharide and corn peptide: Chitosan oligosaccharide with a molecular weight of 1000-3000 (Dalian Zhongke Glek Biotechnology Co., Ltd.) and corn peptide with a molecular weight of less than 1000 (Qiqihar Yixiang Food Co., Ltd.) are dissolved in 100-200 times 70% ethanol at a ratio of (0.5-1.5):1, and the solution is fully dissolved by vortexing; (2) Adjusting the pH of the solution: using 0.4-1.0 mol·L -1Adjust the pH of the mixed chitosan oligosaccharide and corn peptide solution to 9.0-13.0 using NaOH (Tianjin Kaitong Chemical Reagent Co., Ltd.) solution; (3) Maillard reaction to prepare glycosylated corn peptide: Place the above mixed solution in a 60-90℃ water bath for 60-120 min, cool to room temperature after the reaction, add deionized water to the original volume, adjust the pH of the solution to 7.5-8.0, and magnetically stir for 10-30 min. min, to obtain glycosylated corn peptide solution; (4) Glycosylated corn peptide and lutein loading: add an equal volume of lutein (purity ≥90%, Shanghai Ruji Biotechnology Co., Ltd.) standard solution with a concentration of 100 μg / mL to the above glycosylated corn peptide solution, and hydrate for 10-30 min at a temperature of 40-50℃; (5) Preparation of lutein nanoparticles: remove ethanol from the above glycosylated corn peptide-lutein solution by rotary evaporation at a vacuum of -0.08 to -0.1 MPa and a temperature of 40-50℃, and glycosylated corn peptide loaded with lutein nanoparticles (GCPT-LUT) can be obtained.

[0012] Specific Implementation Method Two: The difference between this implementation method and Specific Implementation Method One is that in step (1), chitosan oligosaccharide and corn peptide are dissolved in 100 times the amount of 70% ethanol at a ratio of 1:1. The other steps are the same as in Specific Implementation Method One.

[0013] Specific Implementation Method 3: The difference between this implementation method and Specific Implementation Method 1 is that in step (2), the pH value of the mixed chitosan oligosaccharide and corn peptide solution is adjusted to 12.0. The other steps are the same as in Specific Implementation Method 1.

[0014] Specific Implementation Method Four: The difference between this implementation method and Specific Implementation Method One is that in step (3), the above mixed solution is placed in an 80°C water bath for 100 minutes. The other steps are the same as in Specific Implementation Method One.

[0015] Specific Implementation Method 5: The difference between this implementation method and Specific Implementation Method 1 is that step (4) involves hydration at a temperature of 45°C for 30 minutes. The other steps are the same as in Specific Implementation Method 1.

[0016] Specific Implementation Method Six: The difference between this implementation method and Specific Implementation Method One is that in step (5), the above-mentioned glycosylated corn peptide-lutein hydrated solution is subjected to rotary evaporation at a vacuum of -0.09 MPa and a temperature of 50°C to remove ethanol, thereby obtaining a GCPT-LUT nanoparticle solution. The other steps are the same as in Specific Implementation Method One.

[0017] Specific Implementation Method Seven: The average particle size, potential, and polydispersity index of the Maillard-reactive glycosylated corn peptide-loaded lutein nanoparticles obtained in step (5) of Example 6 were determined respectively. The specific determination method is as follows: COS, GCPT-LUT, and CPT nanoparticle samples were dispersed in deionized water (0.1 mg / mL) and placed in the sample cell of a Malvern Zetasizer Nano ZS90 zeta potential analyzer for analysis. The zeta potential, average particle size, and polydispersity index of the nanoparticles were measured. All measurements were repeated three times. The results are as follows: Figure 1-3 As shown: ① Average particle size analysis of nanoparticles: Particle size analysis results for different samples are as follows Figure 1 As shown, the average particle size of corn peptide (CPT) was 190±7.04 nm, the average particle size of chitosan oligosaccharide (COS) was 164±8.50 nm, the average particle size of corn peptide lutein nanoparticles (CPT-LUT) was 309 nm, and the average particle size of glycosylated corn peptide lutein nanoparticles was 396±5.18 nm (GCPT-LUT). This indicates that the average particle size of glycosylated corn peptide lutein nanoparticles is increased, the distribution is uniform, and the encapsulation effect of lutein is good. ② Determination of polydispersity index (PDI) of nanoparticles: as shown in the figure. Figure 2 As shown, the polydispersity index (PDI = 0.149) of Maillard-glycosylated corn peptide-lutein nanoparticles was lower than that of corn peptide (PDI = 0.304), chitosan oligosaccharide (PDI = 0.253), and corn peptide-lutein nanoparticles (PDI = 0.244), indicating that all three were in a stable monodisperse state, and the lutein nanoparticles encapsulated with Maillard-glycosylated corn peptides had good dispersion stability. ③ Zeta potential measurement of nanoparticles: Figure 3 As shown, the absolute values ​​of the zeta potentials for corn peptide and chitosan oligosaccharide are 33.1 mV and 20 mV, respectively. The zeta potential of glycosylated corn peptide lutein becomes 35.3 mV, and the absolute value of the zeta potential is greater than 30 mV, indicating that the modified nanoparticles have good stability.

[0018] Specific Implementation Method 8: The microstructure of the Maillard-reacted glycosylated corn peptide-loaded lutein nanoparticles obtained in step (5) of Example 6 was analyzed by TEM. The specific method is as follows: GCPT-LUT solution was dropped onto a plasma-treated copper mesh support, dried under an infrared lamp, and the microstructure of the nanoparticles was observed under a transmission electron microscope. The results are as follows: Figure 4 As shown, the Maillard-glycosylated corn peptide lutein nanoparticles are uniformly distributed, with a consistent morphology and structure, and a small average particle size. This result is consistent with the average particle size analysis results, indicating that the corn peptide encapsulated with lutein by Maillard-glycosylated chitosan oligosaccharide forms a nanosystem with good dispersibility and stability.

[0019] Specific Implementation Method Nine: Infrared spectroscopy analysis was performed on the Maillard reaction glycosylated corn peptide-loaded lutein nanoparticles obtained in step (5) of Example 6. The specific method is as follows: The LUT, COS, CPT, and GCPT-LUT samples were dried using a vacuum freeze dryer. The dried samples were mixed with a certain proportion of potassium bromide and pressed, and Fourier transform infrared spectroscopy (FTIR) analysis was performed at 4000-5000 cm⁻¹. −1 Infrared spectra were obtained by recording data within a certain wavenumber range. The results are as follows: Figure 5 As shown: The FTIR spectrum of lutein at 2848 cm⁻¹ -1 and 2921cm -1 There is a characteristic peak at 1670 cm⁻¹, belonging to the specific stretching of -CH₂- and -CH₃- groups, while these two peaks disappear in GCPT-LUT. Corn peptides have two typical amide peaks at 1670 cm⁻¹. -1 Represents C=O stretching, 1452 cm -1 This represents the HN bend. In the GCPT-LUT, it is located at 1670 cm. -1 The peak shifted to 1646 cm. -1 And located at 1452 cm -1 The disappearance of the peak indicates that the corn peptide may bind to lutein through electrostatic and hydrophobic interactions. The FTIR spectrum of the GCPT-LUT is very similar to that of the carrier and has significant COS and lutein characteristic peaks, indicating that lutein is encapsulated within the Maillard-modified corn peptide to form a nanocomposite structure.

Claims

1. A method for preparing Maillard reaction modified corn peptide-loaded lutein nanoparticles, characterized in that: The method includes the following steps: (1) Preparation of chitosan oligosaccharide and corn peptide solution: Chitosan oligosaccharide and corn peptide were dissolved in 70% ethanol in proportion and vortexed to dissolve completely; (2) Adjust the pH of the solution: use 0.4-1.0 mol·L -1 The pH of the mixed chitosan oligosaccharide and corn peptide solution was adjusted to alkaline using NaOH solution. (3) Maillard reaction for preparation of glycosylated corn peptides: The above mixed solution was placed in a water bath at 60-90℃ for reaction. After the reaction was completed, it was cooled to room temperature, and deionized water was added to make up the original volume. The pH value of the solution was adjusted to 7.5-8.0, and the solution was magnetically stirred for 10-30 min to obtain a glycosylated corn peptide solution; (4) Glycosylated corn peptides are loaded with lutein: An equal volume of lutein standard solution with a concentration of 100 μg / mL is added to the above glycosylated corn peptide solution for hydration reaction; (5) Preparation of lutein nanoparticles: The above glycosylated corn peptide-lutein hydrated solution was concentrated under vacuum to remove ethanol, and Maillard glycosylated corn peptide-loaded lutein nanoparticles (GCPT-LUT) were obtained.

2. The method for preparing Maillard reaction modified corn peptide-loaded lutein nanoparticles according to claim 1, characterized in that, The nanoparticles are prepared from corn peptides using Maillard reaction as a carrier. The average particle size is mainly concentrated in the range of 200-400 nm. The loading rate of lutein can reach 91.5%, and the polydispersity index (PDI) is less than 0.15, indicating good dispersibility and stability.

3. The method for preparing Maillard reaction modified corn peptide-loaded lutein nanoparticles according to claim 1, characterized in that, In step (1), chitosan oligosaccharide with a molecular weight of 1000-3000 and corn peptide with a molecular weight of less than 1000 are dissolved in 100-200 times the amount of 70% ethanol at a ratio of (0.5-1.5):

1.

4. The method for preparing Maillard reaction modified corn peptide-loaded lutein nanoparticles according to claim 1, characterized in that, In step (2), the pH of the mixed chitosan oligosaccharide and corn peptide solution is adjusted to 9.0-13.

0.

5. The method for preparing Maillard reaction modified corn peptide-loaded lutein nanoparticles according to claim 1, characterized in that, Step (3) involves placing the above mixed solution in a water bath at 60-90℃ for 60-120 min.

6. The method for preparing Maillard reaction modified corn peptide-loaded lutein nanoparticles according to claim 1, characterized in that, The hydration reaction described in step (4) is carried out at a temperature of 40-50℃ for 10-30 min.

7. The method for preparing Maillard reaction modified corn peptide-loaded lutein nanoparticles according to claim 1, characterized in that, In step (5), the glycosylated corn peptide-lutein hydrated solution is concentrated by rotary evaporation at a vacuum of -0.08 to -0.1 MPa and a temperature of 40-50°C to remove ethanol.

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